Abstract: The extent and nature of the ordering of collagen fibers within a tumor has significant influence on metastasis: in murine breast tumor models, tumor cells move towards blood vessels along fibers that are visible via second harmonic generation (SHG), and SHG is exquisitely sensitive to molecular ordering. Tumor cells that move along SHG fibers are significantly faster than those moving independently, and SHGassociated motility is correlated with metastatic ability. Furthermore, the tumor-host interface contains radially oriented SHG fibers associated with tumor cells invading the surrounding tissue. Lastly, we have shown that treatment of tumors with relaxin, known to alter metastatic ability, alters collagen ordering as detectable by SHG. Consequently, we believe that the process of establishing ordered fibers offers an exciting, and currently unexploited, therapeutic target. To take advantage of this, we must first learn the cellular players and molecular signals by which collagen ordering is induced. Therefore, in this application we propose to determine the key cells and signals which influence the ordering of collagen in breast tumors. The tumor draining lymph node is the first bridgehead for many metastasizing tumor cells and we have exciting preliminary data suggesting that changes in collagen ordering within the node are evident (via SHG) before clinical detection of metastatic cancer, therefore we will also determine the key cells and signals which influence the ordering of collagen in the draining lymph node. Additionally, we will determine if SHG measures of collagen ordering in breast tumors and draining nodes are clinically useful predictors of metastatic outcome in breast cancer patient biopsies. This project has a high impact because it has two independent pathways to clinical relevance, by developing promising antimetastatic drug targets, and by developing an optical method to predict metastatic ability. Public Health Relevance: This project can positively impact public health in several ways. One part of the project is a determination of the cells and signals responsible for ordering collagen in breast tumors, and the resultant effects on metastasis. This will lead to target molecules for therapeutic intervention with the goal of inhibiting breast tumor metastasis. A second part of the project is an evaluation of the ability of optical measures of collagen ordering to predict metastatic ability. This will lead to an improvement in quality of life for breast cancer patients as therapy is customized to patient need.